Compositions for inhibiting oxidation

ABSTRACT

Anthocyanin containing fractions isolated from tart cherries and exhibiting inhibition of oxidation when introduced into various materials is described. The fractions are particularly useful in high and low moisture foods.

This application is a divisional of application Ser. No. 08/799,788filed on Feb. 12, 1997.

BACKGROUND OF THE INVENTION

(1) Summary of the Invention

The present invention relates to a method and compositions forinhibiting oxidation in various materials, particularly lipids and mostparticularly in foods. In particular, the present invention uses a tartcherry natural extract containing a mixture of anthocyanins toaccomplish the inhibition.

(2) Description of Related Art

Prunus Cerasus L. (Rosacease), cv. MONTMORENCY is the major tart cherrycommercially grown in the United States. An artificial red dye isfrequently added to MONTMORENCY cherry food products to enhance its lownatural red color. In order to challenge the MONTMORENCY monoculture, anew cultivar, BALATON tart cherry (Ujferbertoi furtos), was introducedinto the United States in 1984, and has been tested in Michigan, Utah,and Wisconsin. BALATON produces fruits darker than MONTMORENCY.

Colorants like anthocyanins were regarded as the index of quality intart cherries. Most importantly, recent results showed that anthocyaninssuch as cyanidin-3-glucoside have strong antioxidant activities (Tsuda,T., et al, J. Agric. Food Chem. 42:2407-2410 (1994)). The addition ofantioxidants is one of the popular methods to increase the shelf life offood products which is thought to be associated with lipid peroxidation.Natural antioxidants may play an important role in the prevention ofcarcinogenesis. Dietary antioxidants may be effective against thepreoxidative damage in living systems (Halliwell, B. and J. M. C.Gutteridge, Free radicals in biology and medicine. Oxford UniversityPress, New York 416-494 (1989); Osawa, T., et al, Role of dietaryantioxidants in protection against oxidative damage. In antimutagenesisand anticarcinogenesis Mechanisms; Kuroda, Y.; Shankel, D. M., Waters,M. D., Eds.; Plenum Publishing. New York 139-153 (1990)).

Early studies have showed that MONTMORENCY cherry containscyanidin-3-gentiobioside and cyanidin-3-rutinoside (Li, K. C., et al.,J. Am. Chem. Soc. 78:979-980 (1956)). Cyanidin-3-glucosylrutinoside wasalso found in six out of the seven sour cherry varieties (Harborne, J.B., et al., Phytochemistry 3:453-463 (1964)). Dekazos (Dekazos, E.D., J.Food Sci. 35:237-241 (1970)) reported anthocyanin pigments inMONTMORENCY cherry as peonidin-3-rutinoside, peonidin and cyanidin alongwithcyanidin-3-sophoroside, cyanidin-3-rutinoside andcyanidin-3-glucoside. However, cyanidin-3-glucosylrutinoside as well ascyanidin-3-glucoside, cyanidin-3-sophoroside and cyanidin-3-rutinosidewere identified as main pigments in sour cherries. Using HPLC retentionvalues, Chandra et al (Chandra, A., et al., J. Agric. Food Chem.40:967-969 (1992)) reported that cyanidin-3-sophoroside andcyanidin-3-glucoside were the major and minor anthocyanins,respectively, in Michigan grown MONTMORENCY cherry. Similarly,cyanidin-3-xylosylrutinoside was detected as a minor pigment inMONTMORENCY cherry (Shrikhande, A. J. and F. J. Francis, J. Food Sci.38:649-651 (1973)).

U.S. Pat. No. 5,503,867 to Pleva describes the use of whole groundcherries and oat bran in ground meat. The amount of cherries used is 10to 15% by weight and the oat bran is believed to be added to compensatefor the juice in the cherries. In any event, the cherries definitelycontribute a flavor to the meat.

There is a need for natural antioxidants for use, particularly in foodsand other materials containing oxidizable compounds. Lipids in meats areparticularly prone to oxidation and contribute to rancidity in cooked oruncooked foods and natural lipid products for a variety of uses.

OBJECTS

It is therefore an object of the present invention to provide anisolated and purified natural source composition which can be used invarious materials prone to oxidation to prevent the oxidation. Further,it is an object of the present invention to provide a natural sourcecomposition which can be used in foods. Finally, it is an object of thepresent invention to provide a natural source composition which iseconomical to prepare and easy to use. These and other objects willbecome increasingly apparent by reference to the following descriptionand the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the isolated anthocyanins from BALATON andMONTMORENCY cherries. The aglycone cyanidin has a hydroxyl group atposition 3.

FIG. 2 is a schematic flow diagram showing a method of extraction ofcompounds from BALATON cherry.

FIG. 3 is a graph showing the percent inhibition of oxidation by variouscrude cherry extracts produced by the method of FIG. 2. The assay is thefluorescent assay described in Example 2.

FIG. 4 is a schematic flow diagram showing the method of purification ofthe ethyl acetate extract of FIG. 2.

FIG. 5 is a graph showing the percent inhibition of oxidation by thepurified extracts of FIG. 4 using the fluorescent assay.

FIG. 6 is a graph showing the percent inhibition of FIG. 5 in bar graphform.

FIG. 7 is a schematic flow diagram showing the method of purification ofthe methanol fraction of FIG. 4.

FIG. 8 is a graph showing the inhibition of oxidation by the variouspurified fractions produced by the method of FIG. 7.

FIG. 9 is a bar graph showing the relative inhibition of oxidation bythe various fractions of FIG. 7.

FIG. 10 is a graph showing the inhibition of oxidation by caffeic acidwhich is also extracted from the cherries as shown in FIG. 7.

FIG. 11 is a schematic flow diagram of the method of purification of themethanol Fraction of FIG. 2.

FIG. 12 is a graph showing the relative inhibition of oxidation of thevarious purified fractions.

FIG. 13 is a bar graph showing the inhibition of oxidation by thevarious fractions of FIGS. 4 and 11.

FIG. 14 is a schematic flow diagram of the method of extraction ofvarious fractions from BALATON cherry.

FIG. 15 is a graph showing the percentage inhibition of oxidation byvarious fractions of BALATON cherry.

FIG. 16 is a flow diagram showing the method of isolation of variousfractions of MONTMORENCY cherries.

FIG. 17 is a graph showing the inhibition of oxidation by the variousfractions of FIG. 16.

FIG. 18 is a schematic flow diagram showing the method for thepurification of anthocyanins 1, 2, and 3 shown in FIG. 1.

FIG. 19 is a graph showing the inhibition of oxidation by the variousanthocyanin fractions shown in FIG. 18.

FIG. 20 is a graph showing the antioxidant properties of various knownantioxidants.

FIG. 21 is a bar graph showing the inhibiting activity of theanthocyanins compared to various known antioxidants.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a method for inhibiting oxidation of anoxidizable material over time which comprises introducing with thematerial an isolated and purified composition selected from the groupconsisting of anthocyanins, cyanidin and mixtures thereof from a tartcherry so that oxidation of the material is inhibited. Preferably thematerial is a food.

Further, the present invention relates to a product which comprises inadmixture: an isolated and purified composition selected from the groupconsisting of anthocyanins, cyanidin and mixtures thereof from a tartcherry; and a particulate edible bulking agent in an amount betweenabout 0.1 to 30 parts per part of the mixture, which product whenintroduced with an oxidizable material inhibits the oxidation of thematerial.

The term "meat" means pork, chicken, fish, turkey and various othersources of flesh which may be eaten. The meat or other food may be freshand uncooked, cured or cooked, for instance. The term "bulking agent" isused to mean a composition which is added to increase the volume of thecomposition of the purified composition from the tart cherry. Theseinclude any edible starch containing material, protein such as non-fatdry milk. Within this group are flour, sugar, soybean meal, maltodextrinand various condiments, such as salt, pepper, spices and herbs, forinstance. The bulking agent is used in an amount between about 10⁻⁶ and10⁶ parts by weight of the mixture.

The composition is introduced into the food in an amount between about0.001 and 300 mg/gm of the mateeial, particularly a food. The amount isselected so as to not affect the taste of the food and to produce theinhibition of oxidation. The food can be high (wet) or low moisture(dry) as is well known to those skilled in the art.

The anthocyanins and cyanins are isolated from the tart cherries,preferably the BALATON cherries which contained much more of thesecompounds. The ethyl acetate soluble fraction of an aqueous extract ofthe cherries is the most effective, even when compared to more preferredextracts. This is shown from the data in Example 2 and FIGS. 2 to 21.

The BALATON cherry was compared with MONTMORENCY cherry for theanthocyanin content in the following Examples. The results indicate thatboth cherries contain identical anthocyanins. However, BALATON containsabout six times more anthocyanins than in MONTMORENCY. Also, hydrolysisof the total anthocyanins and subsequent GC and NMR experiments with theresulting products indicated that both varieties contain only oneaglycone, cyanidin. Cyanidin and peonidin glycosides are reportedlypresent in MONTMORENCY cherry. The Examples show that the anthocyaninsin BALATON and MONTMORENCY cherries arecyanidin-3-(2"-O-β-D-glucopyranosyl-6"-O-α-L-rhamnopyranosyl-.beta.-D-glucopyranoside),cyanidin-3-(6"-O-α-L-rhamnopyranosyl-β-D-glucopyranoside andcyanidin-3-O-β-D-glucopyranoside as set forth in FIG. 1.

EXAMPLE 1 MATERIALS AND METHODS

Cherry fruits. MONTMORENCY and BALATON tart cherries were obtained fromcommercial growers in Michigan and provided by Cherry MarketingInstitute, Inc. (CMI). The cherries were flushed with nitrogen infreezer bags prior to their storage at -20° C.

General experimental. ¹ H NMR, ¹³ C NMR and DQF COSY spectra wererecorded on a Varian 500 and 300 MHz spectrometers using CD₃ OD/DClsolution at 25° C. All chemical shifts are given in ppm relative to CD₃OD (3.32 ppm). GC was preformed on an HP 5890 II (Hewlett Packard) usingDB-17 (30 m×0.25 mm×0.25 μm) column. The temperature program used was:150° C., initial temperature held for 5 minutes, and then increased to210° C. at 5° C. min⁻¹, maintained for 5 minutes, and finally to 270° C.at 5° C. min⁻¹. The injection port temperature was maintained at 250°C.; The flame ionization detector temperature was 300° C. and carriergas was helium at a linear flow velocity of 4 cm s⁻¹ with a 1:70 splitratio. FAB-MS was carried out on a double focusing mass spectrometerusing Xe as reactant gas in glycerol matrix.

HPLC Conditions for anthocyanin analysis. All samples (20 μl each) wereanalyzed on Chemcopak and Capcellpak C-18 columns (10×250 mm, 5 μm)(Dychrom; Sunnyvale, Calif.) The mobile phase (4% aqueous H₃ PO₄ /CH₃ CN(80:20 v/v) was used under isocratic conditions at a flow rate of 1.5 mlmin⁻¹. The peaks were detected at 520 nm using a Waters PDA detector.Anthocyanins 1-3, 0.5 mg each, were weighed and dissolved in 1 ml of H₂O/CH₃ CN (1:1). The solutions were prepared by the serial dilution ofthe respective stock solutions to afford 0.25, 0.20, 0.10, 0.05, 0.025and 0.0125 mg/ml concentrations, respectively. Quantification ofanthocyanins were carried out using millennium 2010 chromatographymanager.

HPLC Analysis of anthocyanins in cherries. Pitted cherries (100 g) werehomogenized and centrifuged as described above. The supernatant wasdecanted and adjusted with H₂ O to a final volume of 250 ml in avolumetric flask. An aliquot of 1 ml of this solution was passed througha preconditioned C-18 Sep-Pak cartridge (Waters Associates). Theadsorbed pigments were then washed with water (2 ml) followed by H₂O/CH₃ CN (1:1, 1 ml). The eluate was stored at -20° C. prior to HPLCanalysis. Both BALATON and MONTMORENCY showed identical HPLC profile(FIG. 1).

Isolation of crude anthocyanins from tart cherries. The pitted cherries(400 g each of BALATON and MONTMORENCY) were homogenized separately for10 minutes using a Kinematica CH-6010 (Kriens-LU) homogenizer andcentrifuged (Model RC5C, Sorvall Instruments) at 10000 g for 10 minutesat 4° C. to separate insoluble materials from the supernatant. Thesupernatant (400 ml each) was applied to XAD-2 (100 g, amberlite resin,mesh size 20-50; Sigma Chemical Co.) column which was prepared asdescribed by Chandra et al (Chandra, A., et al., J. Agric. Food Chem.41:1062-1065 (1993)). The column was washed with H₂ O (9 L) until thecolorless washings gave a pH of about 7. The adsorbed pigments were theneluted with methanol (500 ml). The red methanolic solution wasconcentrated at 50° C. in vacuo, and the aqueous solution waslyophilized to yield an amorphous red anthocyanin powder, 0.86 and 0.54g, respectively, for BALATON and MONTMORENCY.

Purification of anthocyanins 1-3. The crude anthocyanins from BALATONwas fractionated by C-18 MPLC to produce pure anthocyanins. Theanthocyanin mixture (350 mg) was dissolved in water (2 ml), injectedinto the C-18 column (40×500 mm) and eluted with 4% H₃ PO₄ :CH₃ CN(80:20). Four fractions, I: 125 ml, II: 100 ml, III: 100 ml and IV: 275ml, were collected and evaporated under reduced pressure. The H₃ PO₄from these fractions was removed by passing each fraction throughpreconditioned C-18 Sep-Pak (Waters Associates) with methanol andfollowed by 10% methanol. The adsorbed pigment was washed with 5 mlwater to remove the acids and then eluted with H₂ O/methanol (1:1, 5 ml)to afford pure anthocyanins. The yield of anthocyanins from fractionsI-IV were 53, 24, 133 and 64 mg, respectively. HPLC analysis of thesefractions revealed that fraction I was pure and contained onlyanthocyanin 1. Fraction II contained anthocyanins 1 and 2, fraction IIIhad anthocyanins 2 and 3 and fraction IV contained anthocyanin 3 withother phenolics as indicated in their HPLC profiles.

Since fractions II and III from MPLC contained all three of theanthocyanins, 40 mg of II and 30 mg of III were purified further by HPLCon Capcellpak C-18 column (10×250 mm, 5 μm) (Dychrom; Sunnyvale, Calif.)to yield pure anthocyanins 2 and 3. Peaks were detected using a PDAdetector at 520 and 283 nm, respectively. The mobile phase (4% aqueousH₃ PO₄ :CH₃ CN::83:17 v/v) was used under isocratic conditions at a flowrate of 2.0 ml/min. Respective anthocyanin fractions from HPLCpurification from fractions II and III were combined, dried underreduced pressure and purified further using C-18 Sep-Pak to remove H₃PO₄. The weights of pure anthocyanins 1-3 were 5.7, 8.9 and 2.9 mg,respectively.

Crude anthocyanins from MONTMORENCY (500 mg) was also fractionated byC-18 MPLC as in the case of BALATON. Three bands with red color werecollected as fractions I (10 mg), II (30 mg) and III (20 mg) Fraction Iwas pure and contained anthocyanin 1. Fractions II and III were not pureby HPLC analysis and contained anthocyanins 1-3.

Anthocyaninl:Cyanidin-3-(2"-O-β-D-glucopyranosyl-6"-O-α-L-rhamnosyl-β-D-glucopyranoside.Red amorphous powder; ¹ H NMR (CD₃ OD): δ 1.14 (3H, d, J=6.18, H-6""),2.92 (1H, dt. J=9.28, 3.97 H-5'"), 3.19 (1H, d, J=9.08, 7.74, H-2'"),3.23 (1H, t, J=9.28, H-4'"), 3.27 (1H, dd, J=9.28, 9.50, H-4""), 3.33(1H, dd, J=9.08, 9.28, H-3'"), 3.44 (2H, d, J=3.97, H-6'"), 3.50 (1H,dd, J=9.53, 9.28, H-4") 3.56 (1H, dd, J-9.28, 6.18, H-5""), 3.60 (1H,dd, J=9.50, 3.31, H-3""), 3.61 (1H, dd, J=12.2, 1.76, H-6b"), 3.72 (1H,ddd, J=9.53, 6.41, 1.76 H-5"), 3.77 (1H, dd, J=9.28, 9.08 H-3"), 3.78(1H, dd, J=3.31, 1.54, H-2""), 4.04 (dd, J=12.2, 6.41, H-6a"), 4.05 (1H,dd, J=9.08, 7.29, H-2"), 4.65 (1H, d, J=1.54, H-1""), 4.76 (1H, d,J=7.74, H-1'"), 5.43 (1H, d, J=7.29, H=1"), 6.67 (1H, d, J=1.96, H-6),6.90 (1H, d, J=1.96, H-3), 7.06 (1H, d, J=8.66, H-5'), 8.00 (1H, d,J=2.24 H-2'), 8.18 (1H, dd, J=8.66, 2.24, H-6'), 8.89 (1H, s, H-4); ¹³ CNMR (CD₃ OD): δ 17.9 (C-6""), 62.3 (C-6'"), 67.6 (C-6"), 69.8 (C-5""),70.8 (C-4'"), 71.2 (C-4"), 71.8 (C-2""), 72.4 (C-3""), 73.9 (C-4""),75.9 (C-2'"), 77.2 (C-3"), 77.7 (C-3'"), 77.7 (C-5'"), 77.9 (C-5 "),82.3 (C-2"), 95.2 (C-8, 101.9 (C-1'"), 102.2 (C-1""), 103.5 (C-6), 104.9(C-1"), 113.2 (C-10), 117.6 (C-5'), 118.6 (C-2'), 121.2 (C-1'), 128.3(C-6'), 136.1 (C-4), 145.2 (C-3), 147.4 (C-3'), 155.7 (C-4'), 157.6(C-9), 159.0 (C-5), 164.3 (C-2), 170.4 (C-7).

Anthocyanin 2:Cyanidin-3-(6"-O-α-L-rhamnopyranosyl-β-D-glucopyranoside). Red amorphouspowder, ¹ H NMR (CD₃ OD): δ 1.15 (3H, d, J=6.14, H-6""), 3.34 (1H, dd,J=9.49, 9.22, H-4"), 3.41 (1H, dd, J=9.49, 9.21, H-4'"), 3.54 (1H, dd,J-9.21, 6.14, H-5'"), 3.55 (1H, dd, J=9.22, 9.06, H-3"), 3.62 (1H, dd,J=11.90, 1.62, H-6b"), 3.63 (1H, dd, J=9.49, 3.35, H-3'"), 3.67 (1H, dd,J-9.06, 7.53, H-2"), 3.71 (1H, m, H-5"), 3.80 (1H, dd, J-3.35, 1.67,H-2'"), 4.05 (1H, dd, J=11.90, 6.31, H-6a"), 4.65 (1H, d, J=1.67,H-1'"), 5.29 (1H, d, J=7.53, H-1"), 6.69 (1H, d, J=1.95, H-6), 6.91 (1H,d, J=1.95, H-8), 7.01 (1H, d, J=8.65, H-5'), 8.02 (1H, d, J=2.23, H-2'),8.27 (1H, dd, J=8.65, 2.23, H-6'), 8.92 (1H, s, H-4); ¹³ C NMR (CD₃ OD):δ 17.9 (C-6'"), 67.8 (C-6"), 69.7 (C-5'"), 71.2 (C-4"), 71.9 (C-2'"),72.4 (C-3'"), 73.9 (C-4'"), 74.7 (C-2"), 77.4 (C-3"), 78.0 (C-5"), 95.3(C-8), 102.1 (C-1'"), 103.5 (C-1"), 103.5 (C-6), 113.2 (C-10), 117.5(C-5'), 118.4 (C-2'), 121.2 (C-1'), 128.4 (C-6'), 136.6 (C-4), 145.6(C-3), 147.4 (C-3'), 155.8 (C-4'), 157.6 (C-9), 159.0 (C-5), 164.3(C-2), 170.4 (C-7).

Anthocyanin 3: Cyanidin-3-β-D-glucopyranoside. Red amorphous powder, ¹ HNMR (CD₃ OD): δ 3.34 (1H, dd, J=9.50, 9.22, H-4"), 3.55 (1H, dd, J=9.22,9.0, H-3"), 3.67 (1H, dd, J=9.0, 7.5, H-2"), 3.68 (1H, dd, J=11.90,1.62, H-6b"), 3.71 (1H, m, H-5"), 3.91 (1H, dd, J=11.90, 6.30, H-6a'),5.40 (1H, d, J=7.5, H-1"), 6.71 (1H, d, J=1.95, H-6), 6.98 (1H, d,J=1.95, H-8), 7.07 (1H, d, J=8.65, H-5'), 8.05 (1H, d, J=2.23 H-2'),8.29 (1H, d, J=8.65, 2.23, H-6'), 8.98 (1H, s, H-4). ¹³ C-NMR on pureanthocyanin 3 was not performed due to its low yield.

Cyanidin, the aglycone.

The crude anthocyanin powder from BALATON (55 mg) was hydrolyzed with 3M HCl (15 ml) for 1 hour at 100° C. The red solution was cooled to roomtemperature and stirred with butanol (20 ml). The mixture was extractedwith water (3×20 ml) and the combined water extracts were evaporated todryness at reduced pressure to yield the sugars (30 mg). The red butanollayer was evaporated to dryness (24.3 mg) and the residue was purifiedby silica gel preparative TLC using the solvent system ethyl acetate:formic acid: 2 M HCl::85:6:9. The single red band at R_(f) 0.28 waseluted with MeOH, evaporated under reduced pressure and afforded a redamorphous powder, cyanidin (11.2 mg). Similarly, pure anthocyanins (0.5mg each) were hydrolyzed also to obtain their respective sugars for GCanalysis. The aglycones from anthocyanins 1-3 and the crude anthocyaningave identical R_(f) values and HPLC retention times. Also, allaglycones showed identical ¹ H- and ¹³ C-NMR spectra. ¹ H NMR (CD₃ OD):δ 6.65 (1H, d, J=1.95, H-6), 6.90 (1H, d, J=1.95, H-8), 7.02 (1H, d,J=8.66, H-5'), 8.11 (1H, d, J=2.23, H-2'), 8.17 (1H, dd, J=8.66, 2.23,H-6'), 8.62 (1H, s, H-4); ¹³ C NMR (CD₃ OD): δ 94.7 (C-8), 103.0 (C-6),113.5 (C-10), 117.2 (C-5'), 117.9 (C-2'), 121.9 (C-1'), 127.1 (C-6'),134.0 (C-4), 146.5 (C-3), 147.3 (C-3'), 155.1 (C-4'), 156.9 (C-9), 157.9(C-5), 162.6 (C-2), 168.8 (C-7).

Characterization of sugars by GC analysis. The sugar standards, (1 mgeach) rhamnose, fructose, galactose, glucose and the internal standardPhenyl-δ-D-Glucoside (E. Merck, Darmstadt) and the sugars (1 mg each)obtained from the hydrolysis of crude and pure anthocyanins (1-3) werereacted separately with 30 mg/ml Hydroxylamine HCl in dry pyridine (2ml). The resulting oximes were then reacted with 1.0 mlhexamethyldisilazane (HMDS) and 0.1 ml trifluoroacetic acid (TFA) toyield their silyl derivatives. The samples were then analyzed by GCusing an autosampler. Sugars from anthocyanins 1, 2 and 3 wereidentified by comparing with the retention times of sugar standards. Theretention times were 6.97, 9.83, 10.58, 10.90, 19.82 minutes,respectively, for rhamnose, fructose, galactose, glucose andPhenyl-β-D-Glucoside. The GC analysis of sugars yielded from thehydrolysis of anthocyanins showed that anthocyanin 1 contained a 2:1ratio of glucose and rhamnose. Anthocyanin 2 showed a 1:1 ratio ofglucose and rhamnose and anthocyanin 3 had only glucose. Similarly, theGC analysis of sugars from the crude anthocyanin powder indicated thatit contained only rhamnose and glucose at a ratio of 2:4, respectively.

RESULTS AND DISCUSSION

Lyophilization of 100 g each of BALATON and MONTMORENCY cherriesafforded 8.6 and 7.3% of dry weights, respectively. Sugars and acids inBALATON are about 50% more than that in MONTMORENCY cherries (data notpresented). Similarly, total anthocyanins in BALATON cherry is about sixtimes higher than that in MONTMORENCY cherry based on anthocyaninconcentrations in fractions obtained from MPLC and HPLC purifications.

Prior to the isolation of anthocyanins for spectral characterization,both BALATON and MONTMORENCY cherries were analyzed by HPLC underidentical conditions. HPLC profiles of the cherry extract showed thatthere are two major and one minor anthocyanins in both varieties asindicated by retention times 8.76, 10.58, 13.38 minutes, respectively,for anthocyanins 1-3 (FIG. 1). Also, it was evident from the markeddifference in the red color between these two cherries and HPLC profile(FIG. 1) that MONTMORENCY contained relatively small amounts ofanthocyanins compared to BALATON.

Production of pure Anthocyanins (1-3) from BALATON and MONTMORENCYcherry juices were carried out first by adsorbing the pigment on anAmberlite XAD-2 column (Chandra, A., et al, J. Agric. Food Chem.41:1062-1065 (1993)). The column was washed with water till the eluantgave a pH of @ 7.0. The adsorbed pigments along with other phenolicswere eluted with MeOH. The resulting crude anthocyanins werefractionated and purified by C-18 MPLC and HPLC, respectively, to affordpure anthocyanins for spectral studies. Purification of 500 mg crudeMONTMORENCY anthocyanins from XAD-2 yielded 60 mg of pure anthocyanins1-3 compared to 391.43 mg from BALATON. This indicates that crudeathocyanins from MONTMORENCY obtained from the XAD-2 contained a higherpercentage of other organic compounds.

The presence of cyanidin and respective sugar moieties in anthocyanins1-3 were confirmed by the comparison of their ¹ H- and ¹³ C-NMR chemicalshifts with the published data (Glaβgen, W. E., et al, Phytochemistry31:1593-1601 (1992)). The relative configuration and nature of thesugars in anthocyanins 1 and 2 were deduced from the vicinal and geminal¹ H--¹ H coupling constants and by DQFCOSY.

The ¹ H NMR spectrum of 1 gave signals for three anomeric protonsappeared at δ 5.43, 4.76 and 4.64, respectively for glucose (attached tothe aglycone), glucose and rhamnose. Also, the presence ofβ-D-glucosidic linkage in 1 was confirmed by the large couplingconstants for the anomeric protons. The signal at δ 4.64 ppmcorresponded to the anomeric proton of an L-rhamnopyranose and the 1.8Hz coupling constant indicated an α-glycosidic linkage.

The ¹³ C NMR chemical shifts observed for anthocyanins in BALATON andMONTMORENCY were similar to the published data (Agrawal, P. K., et al.,Flavonoid glycoside in carbon-13 NMR of flavonoids. (Ed. Agrawal, P. K.)Elsevier, Amsterdam, 283-364 (1989)). The C-7 resonated at very lowfield 170.4 ppm compared to the rest of the oxygenated aromatic carbonsin anthocyanins. The oxygen cation in ring C is responsible for thedownfield shift of C-7 carbon. The ¹³ C NMR signal for C-5 carbon in 1at δ 69.8 confirmed the rhamnosyl moiety with an α-linkage to theglucose (Agrawal, P. K., Phytochemistry 31:3307-3330 (1992)).

The downfield shift of the C-2" proton in 1 relative to the C-2" signalof 2 was due to the glucosylation and indicated a 1,2 linkage betweenthe two glucose units. Similarly, the downfield shift of C-6" proton inthe ¹ H=NMR spectrum of 1 relative to the C-6 proton signal of glucosewas due to the rhamnose moiety and indicated a 1,6 linkage between theglucose and rhamnose. Therefore, anthocyanin 1 is confirmed to becyanidin-3-(2"-O-β-D-glucopyranosyl-6"-O-α-L-rhamnopyranosyl-β-D-glucopyroside).Anthocyanin 1 gave a molecular ion at m/z 758 [M+H]⁺ and the base peakat m/z 596 [M+H-C₆ H₁₀ O₅ ]) in the FAB MS further confirmed thepresence of cyanidin, two glucose and one rhamnose moieties in 1.

¹ H NMR spectrum of 2 showed signals for two anomeric protons at δ 5.29(J=7.53 Hz) and 4.64 (J=1.67). This indicated the presence of aβ-D-glucose because all vicinal coupling constants were 7.53˜11.9 ppm.The doublet (J=6.14 Hz) at 1.15 ppm of a methyl group confirmed one ofthe sugars as rhamnose in 2. The small coupling constant of 1.67 Hz forthe anomeric proton suggested an α-rhamnosyl linkage. The C-6" proton ofglucose at 5.5 ppm indicated a 1,6 linkage between the glucose andrhamnose. The FAB-MS of 2 gave the moelcular ion at m/z 596 [M+H]⁺ andconfirmed its structure ascyanidin-3-(6"-O-α-L-rhamnopyranosyl-β-D-glucopyranoside.

The ¹ H NMR of anthocyanin 3 revealed only a single glucose moietyattached to the aglycone cyanidin. The structure of 3 was confirmed tobe cyanidin-3-β-D-glucoside. Hydrolysis of crude anthocyanins and TLC ofresulting products showed that there was only one aglycone present inboth BALATON and MONTMORENCY cherries. ¹³ C NMR data showed thisaglycone as cyanidin.

The results suggest that there are only three identical anthocyaninspresent in both BALATON and MONTMORENCY cherries. The yields ofspectroscopically pure anthocyanins 1-3 in 100 g of fresh BALATON andMONTMORENCY cherries were 14.99, 6.20; 6.18, 0.97; 2.42, 0.35 mg,respectively. The amount of anthocyanins isolated from MONTMORENCY inour studies show that it is lower than the reported yields (Dekazos, E.D., J. Food Sci. 35:237-241 (1970)). However, this may be due to varyingenvironmental and nutritional factors. An important point to note isthat when anthocyanins are monitored by HPLC at 520 nm, other phenoliccompounds which absorb at 23 nm are ignored. We have isolated at leastfour phenolic compounds co-eluted with the anthocyanins and detected at283 nm.

Chandra et al (J. Agric. Food Chem. 40:967-969 (1992)) reported thatMONTMORENCY cherries grown in Michigan contain onlycyanidin-3-sophoroside and cyanidin-3-glucoside. These results wereconfirmed by matching their retention times to those of the anthocyaninspresent in an authentic sample of blackberry juice described by Hong andWrolstad (Hong, V., et al., J. Agric. Food Chem. 38:698-708 (1990); andHong, V., et al., J. Agric. Food Chem. 38:708-715 (1990)). Also, earlierreports indicated that there are peonidin-3-glycoside andpeonidin-3-galactoside present in MONTMORENCY cherry. However, thepresent invention indicates that MONTMORENCY contains the same number ofanthocyanins found in BALATON cherries and were identical. This is thefirst description of the characterization of anthocyanins from BALATONcherries and the spectral characterization of anthocyanins fromMONTMORENCY cherries.

EXAMPLE 2

The anthocyanin products of Example 1 were tested under variousconditions using a fluorescent assay for antioxidant activity. Thefluorescent assay is described first.

Fluorescence assay for antioxidant activity (general): The need toscreen large numbers of compounds or extracts for antioxidant activityrequires that a model system (or systems) be employed which reasonablywell represents the structural and functional characteristics of thesubstrate in the food product. The test must also be sensitive, rapid,and inexpensive. Our laboratory has developed a fluorescence-based assayfor evaluating antioxidant efficacy which fulfills these criteria(Arora, A., and G. M. Strasburg, J. Am. Chem. Soc. 1996)). Largeunilamellar vesicles consisting of1-stearoyl-2-linoleoly-sn-glycero-3-phosphocholine are prepared, whichclosely resemble the properties of biological membranes, one of theprimary sites of peroxidation. A fluorescent probe,1,6-diphenylhexatriene propionic acid, is incorporated into themembranes such that the polar head group anchors the probe near theaqueous interface, while the hydrophobic portion lies parallel to thefatty acid chains. This probe reacts with the free radicals generatedduring peroxidation, resulting in a decrease in fluorescence intensitywith time. A peroxidation initiator (such as ferrous metal ions or thefree radical generator AAPH (What is AAPH) is used to start thereaction, and the kinetics of fluorescence decrease are determined inthe presence or absence of the antioxidant to be tested. An assay for acompound at a given concentration presently takes only twenty-oneminutes, consumes only a few micrograms of lipid, and can be readilyconducted with a simple fluorometer.

Fluorescence assay for antioxidant activity (specific details): Largeunilamellar vesicles (LUVs) are prepared from1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine according to theprocedure outlined by MacDonald et al (MacDonald, R. C., et al.,Biochim. Biophys. Acta 1061:297-303 (1991)). Briefly, the lipid isdissolved in chloroform, and is dried to a thin film using a rotaryevaporator. The dried film is resuspended in an aqueous buffer, and isrepeatedly extruded through a polycarbonate filter of 100 nm pore sizeusing a Liposofast piposome extruder (Avestin, Inc., Ottawa, Canada).The homogeneity of size (80-100 nm) and the unilamellar nature of thevesicles have been confirmed by our laboratory using freeze-fracturescanning electron microscopy. The fluorescent probe,diphenylhexatriene-propionic acid (DPH-PA), is incorporated into thevesicles during preparation at a mole ratio of 1:350 (probe:lipid). Forthe fluorescence experiments, LUVs containing DPH-PA is suspended at afinal concentration of 100 μM in 100 mM NaCl, 50 mM tris-HEPES buffer atpH 7.0. The fluorescent probe is excited at 384 nm and emission ismonitored at 423 nm. Lipid oxidation is inhibited in the LUVs byaddition of ferrous ions or the free radical generator AAPH; theprogress is monitored by the decrease of the fluorescence intensity ofDPH-PA resulting from reaction with free radicals generated overtwenty-one minutes. A plot of the decrease of fluorescence intensity asa function of time will be used to determine the kinetics of lipidoxidation.

The results of the assays of the various fractions are shown in FIGS. 2to 21 and Table 1. The results show that a mixture of the crudeanthocyanin extract with methylacetate is more effective than any of thepurified extracts inhibiting oxidation.

EXAMPLE 3

Examples of use in foods, particularly meats, to be added.

    ______________________________________                                                           Fl. int.                                                                      at 21    % of                                              % of inhibition of cherry extracts                                                               min..sup.1                                                                             inh..sup.2                                        ______________________________________                                        Blank              0.92     0.934                                             Fe2+               0.28     0.35                                              Bal./hexane (50 ppm)(HW/24/112.1                                                                 0.58             46.9%                                     Mont./hexane (50 ppm)                                                                            0.61             51.6%                                     Bal./EtOAc (25 ppm)(HW/24/112/2)                                                                          0.957   103.9%                                    Mont./EtOAc (25 ppm)        0.936   100.3%                                    Bal./MeOH (25 ppm)(HW/24/112/3)                                                                           0.896   93.5%                                     Mont./MeOH (25 ppm)         0.908   95.5%                                     Bal./Antho. crd (25 ppm)    0.941   101.2%                                    Mont./antho. crd (25 ppm)   0.699   59.8%                                     Blank              0.93                                                       Fe2+               0.24                                                       [HW/24/120/1][YC/63-1](50 ppm)                                                                   0.28             5.8%                                      [HW/24/120/2][YC/63-2](50 ppm)                                                                   0.23             -1.4%                                     [HW/24/120/3][YC/63-3](50 ppm)                                                                   0.34              14.5%                                    [HW/24/120/4][YC/63-4](50 ppm)                                                                   0.63             -56.5%                                    [HW/24/120/5][YC/63-5](50 ppm)                                                                   0.75             73.9%                                     [HW/24/120/6][YC/63-6](50 ppm)                                                                   0.82             84.1%                                     [HW/24/120/7][YC/63-7](50 ppm)                                                                   0.86             89.9%                                     Blank              0.92                                                       Fe2+               0.18                                                       [HW/24/113/1][YC/58-2](50 ppm)                                                                   0.24             8.1%                                      [HW/24/113/2][YC/58-3](50 ppm)                                                                   0.37             25.7%                                     [HW/24/113/3][YC/58-4](50 ppm)                                                                   0.41              31.1%                                    [HW/24/113/4][YC/58-5](50 ppm)                                                                   0.59             55.4%                                     [HW/24/113/1][YC/65-1](50 ppm)                                                                   0.7              66.2%                                     [HW/24/132/2][YC/65-2](50 ppm)                                                                   0.66             61.0%                                     [HW/24/132/3][YC/65-3](50 ppm)                                                                   0.39             26.0%                                     Blank              0.96                                                       Fe2+               0.19                                                       Blank              0.91                                                       Fe2+               0.21                                                       Aglycone (2 μM) 0.61             57.1%                                     Anthocyanin 1 (2 μM)(HW/24/66/a)                                                              0.48             38.6%                                     Anthocyanin II (HW/24/95/3)                                                                      0.7              70.0%                                     Anthocyanin III (2 μM)(HW/24/95/5)                                                            0.74             75.7%                                     a-Tocopherol (2 μM)                                                                           0.21             0.0%                                      BHA (2 μm)      0.82             87.1%                                     BHT (2 μM)      0.87             94.3%                                     Propyl gallate (2 μM)                                                                         0.76             78.6%                                     TBHQ (2 μM)     0.84             90.0%                                     ______________________________________                                         .sup.1 Fuorescent intenity at 21 minutes.                                     .sup.2 Percent Inhibition based upon Fe.sup.+2 oxidation.                

The anthocyanins are naturally occurring and are non-toxic. The ethylacetate extracts are particularly effective.

The results are shown in FIGS. 3, 5, 6, 8, 9, 10, 12, 13, 15, 17, 19, 20and 21.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

We claim:
 1. A product which comprises in admixture:(a) an isolated andpurified composition which consists essentially of a mixture ofanthocyanins which iscyanidin-3-(2"-O-β-D-glucopyranosyl-6"-O-α-L-rhamnosyl-β-D-glucopyranoside),cyanidin-3-(6"-O-α-L-rhamnopyranosyl-β-D-glucopyranoside) andcyanidin-3-β-D-glucopyranoside, from a tart cherry; and (b) aparticulate edible bulking agent in an amount between about 10⁻⁶ to 10⁶parts per part by weight of the mixture, which product when introducedwith an oxidizable material inhibits the oxidation of the material. 2.The composition of claim 1 wherein the bulking agent is an edible starchor protein.
 3. The composition of claim 1 wherein the bulking agent ispowdered non-fat dry milk.
 4. The composition of claim 1 wherein thetart cherry is selected from the group consisting of BALATON andMONTMORENCY.